Method of conducting a wagering game

ABSTRACT

Apparatus and method for enhanced biomass production by electrical waveform shaping are disclosed. The invention relates to cultivating a biological source cell in a liquid-medium bioreactor and applying a waveform regulated electric field potential to the source cell, wherein the liquid medium comprises one or more ionizable components. The waveform that regulates the applied field potential comprises a plurality of modes such that at least one mode orders an applied field potential capable of generating an ion from the ionizable component and at least one mode orders an applied field potential capable of inducing migration of the generated ion within the liquid medium. Related apparatus and methods are also disclosed.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional PatentApplication No. 60/176,717 filed Jan. 14, 2000, which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

[0002] The present invention relates generally to production of biomass,and in particular to a process for generating biomass in a bioreactor byapplying a waveform regulated electric field potential.

BACKGROUND OF THE INVENTION

[0003] Cell growth in bioreactors provides a tool for research andproduction in a wide variety of commercial and industrial contexts,including pharmaceutical, nutritional, chemical, environmental and otherapplications. Depending on the specific biological organism beingcultured, various configurations of bioreactor hardware and softwarehave been devised in efforts to optimize biomass production, forexample, to increase yields of fermentation products or to maximize cellgrowth by maximizing volumetric productivity (g l⁻¹ h⁻¹) by highcell-density cultivation (HCDC). Bioreactors often may comprisecontainers of various shapes, dimensions and materials, such as tanks,chambers, tubes, kettles, flasks or the like fabricated from glass,metal, plastic, polymer, ceramic, membranes including ceramic and/ordialysis membranes or other suitable materials selected according to theparticular organism and process to be used. For a review of HCDC, see,e.g., Riesenberg et al., 1999 Appl. Microbiol. Biotechnol. 51:422-430.

[0004] For a variety of reasons, undesirable settling of cultured cellsor organisms, or accumulation of noxious, toxic or inhibitory productsor by-products in the bioreactor, may impair the efficiency of biomassproduction. For example, settling, aggregation or clumping of growingcell cultures into a slurry or sludge can retard cell growth rates bypreventing access of the cells to microenvironments having an optimalnutrient, gas and/or pH balance. Attempts to mitigate these and relatedproblems among the many bioreactor configurations known to the artinclude redistribution of the bioreactor contents by subjecting thebioreactor to regular, physical movement or by introducing a pump,circulator, agitator, stirrer, mixer, impeller, lift, airlift or otherdevice into the bioreactor for this purpose; introduction ofmicrocarrier particles to provide suspension substrates for cell growth;or sparging the bioreactor by bubbling a suitable gas through the liquidmedium slurry. These and other bioreactor designs known to the art,however, suffer from one or more shortcomings, such as mechanical damageto the cultured cells in the bioreactor, suitability of some but notother specific cells or organisms for propagation in a particularbioreactor configuration, persistent difficulty in maintaining optimalgrowth conditions for a maximum number of cells and other problems. Forexample, limited solubility of nutrients and other solid and gaseoussubstrates in the medium, product instability, degradation orvolatility, increased medium viscosity, inability to maintain optimalmixing in a pseudoplastic fluid such as a liquid medium containing ahigh biomass concentration, high evolution of carbon dioxide and/orheat, and high oxygen demand may all impair volumetric productivity(Riesenberg et al., 1999).

[0005] Clearly there is a need for an improved process and a suitablebioreactor for producing biomass from a wide range of organisms and celltypes under a wide range of conditions. The present invention providesimproved apparatus and methods for generating biomass, includingaccelerated rates of cell growth and proliferation, and enhanced yieldsof biological materials, and offers other related advantages.

SUMMARY OF THE INVENTION

[0006] The present invention provides an apparatus and method forenhanced biomass production through the use of electrical waveformshaping. Accordingly, it is an aspect of the invention to provide amethod for enhancing biomass production in a bioreactor, comprisingproviding a liquid-medium bioreactor that comprises (a) a liquid-mediumcontainment vessel having an axis and comprising a wall and a floor andcontaining a liquid medium, and (b) a first and a second electrodemounted on said wall and mutually opposed along said axis of theliquid-medium containment vessel and immersed in the liquid medium,wherein (i) the first electrode comprises a first electrode surface andthe second electrode comprises a second electrode surface, and saidfirst and second electrode surfaces are substantially parallel to oneanother, and (ii) the first and second electrodes are insulated fromsaid wall and said floor of the containment vessel, wherein said liquidmedium comprises at least one ionizable component; and applying awaveform-regulated electric field potential to at least one biologicalsource cell within the liquid medium in the liquid-medium bioreactor,wherein said waveform regulated electric field potential comprises afirst waveform mode field potential capable of generating at least oneion from the ionizable component when applied to the liquid medium, anda second waveform mode field potential capable of inducing migration ofsaid ion within the liquid medium when said second waveform mode fieldpotential is applied to the liquid medium, and thereby enhancing biomassproduction in the bioreactor.

[0007] In certain embodiments, the liquid-medium containment vesselfurther comprises at least one liquid-medium circulation chamberimmersed in the liquid medium, said circulation chamber being in fluidcommunication with a separate liquid-medium reservoir; (b) at least oneelectrode of the liquid-medium containment vessel is positioned withinthe liquid-medium circulation chamber, said liquid-medium circulationchamber comprising (i) a continuous permeable membrane wall situatedbetween the electrode and the biological source cell, and (ii) a conduitto said separate liquid-medium reservoir; and (c) said separateliquid-medium reservoir comprises (i) a closed tank having liquid mediumtherein, (ii) a circulating means for circulating liquid medium betweenthe liquid-medium circulation chamber and the separate liquid-mediumreservoir, and (iii) an ion trap in fluid communication with theinterior of the tank; whereby circulation of liquid medium between theliquid-medium circulation chamber and the separate liquid-mediumreservoir permits trapping of at least one ion from the liquid medium inthe ion trap. According to certain further embodiments (a) the firstelectrode is positioned within a first liquid-medium circulation chamberthat is in fluid communication with a first separate liquid-mediumreservoir comprising a first ion trap, and (b) the second electrode ispositioned within a second liquid-medium circulation chamber that is influid communication with a second separate liquid-medium reservoircomprising a second ion trap, whereby circulation of liquid mediumbetween the first liquid-medium circulation chamber and the firstseparate liquid-medium reservoir permits trapping of at least one firstspecies of ion from the liquid medium in the first ion trap andcirculation of liquid medium between the second liquid-mediumcirculation chamber and the second separate liquid-medium reservoirpermits trapping of at least one second species of ion from the liquidmedium in the second ion trap.

[0008] In another embodiment the waveform regulated electric fieldpotential is bimodal, and in another embodiment the waveform regulatedelectric field potential comprises at least three modes. In certainembodiments, the biological source is a prokaryote, an archaebacteriumor a eukaryote. In certain further embodiments, the prokaryote isEscherica coli, Staphylococcus aureus, Pseudomonas aeruginosa orBacillus thuringiensis. In other embodiments the eukaryote is a yeast, afungus, a plant, an invertebrate animal or a vertebrate animal. Incertain further embodiments the yeast is Phaffia rhodozyma,Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris,Pichia stipitis, Candida utilis, Candida albicans, Candidaguilliermondii or Cryptococcus albidus. In certain other embodiments,the fungus is Metarhizium flavivoride, Beauvueria bassiana, Paecilomycesfumosoreus or Gladiocladium fimbriatum. In certain other embodiments theplant is Taxus brevifolia. In other embodiments the invertebrate animalis a nematode (e.g., C. elegans) or an insect (e.g., Trichoplusia ni).In other embodiments the vertebrate animal is a reptile, an amphibian, abird, a fish or a mammal. In certain further embodiments the mammal is ahuman, a non-human primate, a rodent, a bovine, an equine, an ovine anda porcine. In another embodiment, the archaebacterium is Marinococcus orSulfolobus shibatae.

[0009] In certain other embodiments the liquid medium is an aqueousmedium. In certain embodiments the liquid medium comprises 1% yeastextract, 2% tryptone peptone and 2% dextrose. In certain embodiments theionizable component is water, and in certain other embodiments theionizable component is an organic molecule having a hydroxyl group. Incertain embodiments the ion is singlet oxygen, Cl⁻, Na⁺, K⁺ or ammonium.

[0010] According to certain embodiments of the present invention, thefirst waveform mode field potential is at least 12.3 volts. According tocertain other embodiments, the second mode field potential is notgreater than one volt.

[0011] These and other aspects of the present invention will becomeapparent upon reference to the following detailed description andattached drawings. All references disclosed herein are herebyincorporated by reference in their entirety as if each was incorporatedindividually.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 shows an example of a liquid-medium bioreactor.

[0013]FIG. 2 shows an example of an electrode for use in a liquid-mediumbioreactor.

[0014]FIG. 3 shows a growth profile of S. cerevisiae cultivated in aliquid-medium bioreactor.

[0015]FIG. 3 depicts enhanced biomass production by applying awaveform-regulated electric field potential to a biological source cellin a liquid medium bioreactor.

[0016]FIG. 4 depicts a waveform for regulating an applied electric fieldpotential.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention pertains in part to the unexpecteddiscovery that remarkable enhancements of biomass production can beachieved in a bioreactor by applying a waveform regulated electric fieldpotential to a liquid medium containing a cell derived from a biologicalsource. The invention therefore provides an improved apparatus andmethod for generating biomass, including accelerated rates of cellgrowth and proliferation, and enhanced yields of biological materials.In particular, the invention relates in part to the surprisingobservation that desirable bioreactor conditions that are favorable formaintenance of a wide variety of biological source cells, andconcomitant augmentation of biomass production, can be effected throughwaveform regulated electric potentials that initiate generation ofspecific gas ions from liquid medium components. The invention thus alsoprovides electrohydrodynamic mixing within a liquid-medium bioreactor ina manner that does not require bubbling gas sparging, airlift devices,agitators or other mechanical mixing strategies.

[0018] Liquid Medium

[0019] A liquid medium for use in a bioreactor according to the presentinvention includes any suitable liquid solution, suspension,formulation, mixture or the like for biological source cell growth thatis in liquid form under the conditions used for enhancing biomassproduction and that comprises at least one ionizable component. Asuitable liquid medium may be selected by a person having ordinary skillin the art based on the particular biological source cell to becultivated, as known in the art and provided herein. Typically, a usefulliquid medium that comprises at least one ionizable component willfurther comprise appropriate solutes, for example, suitable salts and/orsugars to maintain isotonicity with the biological source cell;nutrients as may be required for a particular biological source cell,such as an energy source, a carbon source, a nitrogen source, essentialamino acids, vitamins, cofactors and the like; pH buffering agents; andoptionally additional stabilizers, growth factors, proteins,metabolites, serum or other biological source cell liquid mediumadditives. In particularly preferred embodiments the liquid medium is anaqueous medium, wherein water and/or another medium component may be theionizable component as provided herein. Chemically defined mediarepresent preferred liquid media and have been extensively characterizedfor use with a variety of cell types, including use in bioreactors (forreview, see, e.g., Zhang et al., 1999 Appl. Microbiol. Biotechnol.51:407; see also, e.g., Reddy et al., 1999 Appl. Microbiol. Biotechnol.51:614; Zhang et al. 1996 Appl. Microbiol. Biotechnol. 44:568). Numerousother liquid media that support biological source cell growth (e.g.,cell division) and/or product formation (e.g., cellular gene expression,production or elaboration of desired products, including proteins,enzymes, nucleic acids, lipids, carbohydrates or other biochemicalintermediates, metabolites, catabolites, substrates, precursors,cofactors or the like) are known for many particular biological sourcecells as provided herein, or can be readily determined by routinescreening and without undue experimentation by a person familiar withthe art, based on the present disclosure.

[0020] As noted above, a liquid medium according to the presentinvention comprises at least one ionizable component. In preferredembodiments the liquid medium is an aqueous medium and at least oneionizable component therein is water which, when a waveform regulatedelectric field potential is applied to the liquid medium, may beelectrolytically dissociated to produce monatomic oxygen (O⁻) anion.Without wishing to be bound by theory, according to the presentinvention the first waveform mode (of a waveform having a plurality ofmodes) regulates a first waveform mode field potential (i.e., an initialpulse voltage) that induces ionization of an ionizable component of theliquid medium to generate a desired ion by a process that, by virtue ofthe transient nature of the first waveform mode and as a function of thefull waveform, which may further be a function of the bioreactorcontainment vessel dimensions and/or geometry, is not deleterious tobiological source cells.

[0021] Accordingly, in the embodiment just described, the first waveformmode field potential induces the generation of oxygen anion from theionizable medium component water, but the invention need not be solimited. Depending upon, inter alia, the composition of the liquidmedium, the waveform selected to regulate the application of electricfield potentials to the medium and the particular response properties ofa selected biological source cell to the field potentials ordered by thewaveform under the conditions employed, a wide variety of desired ionicspecies may be generated from an appropriately formulated liquid medium.

[0022] Thus, a liquid medium for use according to the invention maycontain any of a wide variety of ionizable components, the ionization ofwhich may be induced by a waveform regulated electric field potentialapplied to the biological source cell in the liquid medium in abioreactor. Examples of ions so generated that may be useful forenhancing biomass production include monatomic oxygen (O⁻) anion,carbonium (C⁺) ion, ammonium (NW₄ ⁺) ion and other ionic species thatwill depend upon various factors, including the composition of theliquid medium, the bioreactor and electrode configurations and thewaveform employed (all of which may be selected and regulated to producea desired conductivity), as well as other factors. Ionizable componentsmay also include organic molecules, many of which comprise structuresthat are readily amenable to electron gain or loss, to generate ionicspecies. For example, ionizable components that are organic moleculeshaving hydroxyl groups may be present in liquid media according tocertain preferred embodiments of the invention. According tonon-limiting theory, ionizable components including organic moleculesmay but need not provide a source of nutritional or respiratorymetabolites to a growing biomass. For instance, certain ionic speciesthat may be derived from liquid medium ionizable components as providedherein may not enhance food properties of the liquid medium per se, butmay serve an adjunctive mixing role that enhances biomass production.

[0023] Liquid-Medium Bioreactor

[0024] A suitable bioreactor for use according to the present inventionmay be virtually any bioreactor that can contain liquid medium asprovided herein, and that has or can be equipped with first and secondelectrodes, as also described herein. Thus, a liquid medium bioreactorcomprises a liquid-medium containment vessel comprising a wall and afloor and containing a liquid medium, which refers to a container havingany of various shapes, dimensions and materials, such as a tank,chamber, tube, kettle, flask or the like fabricated from glass, metal,plastic, polymer, ceramic, membranes including ceramic and/or dialysismembranes or other suitable materials or combinations of materialsselected according to the particular organism, liquid medium and processto be used.

[0025] For purposes of determining electrode mounting positions, theliquid-medium containment vessel has an axis that may in certainpreferred embodiments be a longitudinal axis, but the invention need notbe so limited, such that symmetrical (e.g., radially symmetrical) orasymmetrical (e.g., irregularly shaped) liquid-medium containmentvessels may also be selected. A first and a second electrode are mountedon the wall of the liquid-medium containment vessel at opposite endsalong the axis, each electrode comprising an electrode surface such thatthe mounted electrode surfaces are immersed in the liquid medium andsubstantially parallel to one another, i.e., a plane defined by thefirst electrode surface is parallel or very nearly parallel to a planedefined by the second electrode surface. For instance, in certainembodiments wherein the liquid medium containment vessel has alongitudinal axis, positioning of the electrodes may be performed in amanner such that waveform-regulated, applied electric field lines may befocused within a semiconductive liquid medium to create a region ofelectromagnetic field focus, such that one or more particular specieswithin the liquid medium accumulate preferentially in that region.According to certain further embodiments, circulation of the liquidmedium in the vicinity of the vessel wall, as described in greaterdetail below, may further promote such preferential accumulation.

[0026] The electrodes are mounted in a manner that insulates them from(i.e.,. impairs conductance of any electric current applied through theelectrodes to) the wall and floor of the liquid-medium containmentvessel and provides delivery of electric field potential substantiallyto the liquid medium, where such insulation may be accomplished by anyof a number of designs with which those having skill in the art will befamiliar, including selection of non-conducting materials for theconstruction of the containment vessel wall and floor. Electrodes may beconstructed from a variety of conductive materials that are known in theart and that may be designed to have the desired size, shape andchemical compatibility with the liquid medium. Examples of electrodematerials include stainless steel, copper, graphite, silver, aluminumand platinum that may, depending on the size and shape of theliquid-containment vessel, be provided as wires, sheets, plates, coils,arrays or other configurations.

[0027] Turning to FIG. 1 there is provided an exemplary pictorialillustration of a liquid-medium bioreactor 1 for use according to thepresent invention. The bioreactor comprises a liquid-medium containmentvessel 2 having an axis 3 and comprising a wall 4 and a floor 5 forcontaining liquid medium. A first electrode 6 in a first liquid-mediumcirculation chamber 7 and a second electrode 6 in a second liquid-mediumcirculation chamber 8 are mounted on the wall and mutually opposed alongthe axis 3. A conduit 9 provides fluid communication to a separateliquid-medium reservoir. FIG. 2A provides an exemplary front perspectivepictorial illustration of an electrode housing 12 mounted in a sectionof liquid-medium containment vessel wall 4 and comprising a perforatedstainless steel electrode surface 6 in electrode communication with astainless steel contact 10 that is in communication to the exterior ofthe bioreactor through an insulated port 11. A reverse perspectivepictorial illustration of the electrode housing 12 is provided in FIG.2B, which shows a permeable membrane 13 that is positioned between theelectrode surface and the biological source cell, thereby forming aliquid-medium circulation chamber 7 that contains the electrode surface.

[0028] In a certain embodiment of the invention, the liquid-mediumcontainment vessel further comprises at least one liquid-mediumcirculation chamber that is immersed in the liquid medium and in fluidcommunication with a separate liquid-medium reservoir. In thisembodiment, at least one electrode is positioned within theliquid-medium circulation chamber, which comprises a continuouspermeable membrane wall situated between the electrode and thebiological source cell and a conduit for the passage of fluid to andfrom the separate liquid-medium reservoir. A variety of suitablematerials may be employed for the permeable membrane wall, depending onthe desired physicochemical properties, which will derive in part fromthe bioreactor conditions to be used (e.g., temperature, pH, appliedfield, etc.) and will in any event be sufficient to exclude thebiological source cell from the circulation chamber. For instance,suitable permeable membranes may include dialysis membranes (e.g.,Portner et al., 1998 Appl. Microbiol. Biotechnol. 50:403); gas permeablemembranes (e.g., U.S. Pat. No. 6,001,642, U.S. Pat. No. 5,763,279 andreferences cited therein); ceramic, metallic, nylon, polymer or fibermembranes, or a membrane made from any other suitable material.

[0029] Accordingly and in certain particularly preferred embodiments, atleast one and preferably two electrodes are separated from thebiological source cell by a continuous permeable membrane wall havingdesired physicochemical properties. For instance, in certain embodimentsa membrane material may be employed that exhibits selective propertieswith regard to the molecular weight and/or charge (or other propertiesincluding those noted below) of solutes (including ionic species)capable of passing through such a membrane. The membrane materialselected to surround the anode may, but need not, be the same as themembrane material selected to surround the cathode. In these and relatedembodiments, the membranes may specifically allow an ion generated froman ionizable medium component as provided herein to pass from thevicinity of a first electrode through the membranes to the vicinity of asecond electrode. Without wishing to be bound by theory, according tosuch embodiments an ionizable component may, as its ionic state isaltered, pass to and from distinct liquid-medium circulation chamberscontaining electrodes of opposite polarities, permitting energy transferto the liquid medium in the form of electrical current (e.g., movementof ions). In this manner the environment of the biological source cellcan be varied, for example, to maintain such cells in a desired level ofone or more particular ionized components (e.g., oxygen). Similarly andin related embodiments, the invention contemplates the use of waveformregulated electric field potentials in a liquid-medium bioreactor toeffect movement of a medium component, of a product of a chemicalmodification of a medium component, or of biomass, includingpreferential migration of a selected portion of biomass (e.g., cells,one or more protein species, other biomass products, etc.), to a desiredposition in the bioreactor.

[0030] As further examples, according to such embodiments a membrane,such as a permeable or semipermeable membrane, may be used that permitsdesirable ionic migration for certain species (e.g., oxygen, ammonium,carbonium, etc.) while excluding from transmembrane passage certainother ionized species (e.g., peptides, proteins, sugars, carbohydrates,etc.). Membranes capable of effecting such selectivity are known tothose having ordinary skill in the art, as also noted above. The use ofsuch membranes affords certain advantages that will be readilyappreciated, such as preventing the undesired depletion from the liquidmedium, during biomass generation in the bioreactor, of specific liquidmedium components (e.g., proteins, sugars, amino acids, carbohydrates,etc.) by limiting the access of such components to the electrodes, orlimiting electrode fouling. Suitable membranes, in addition to thosedescribed elsewhere herein, may include Magna™ nylon transfer membranes(MSI, Westboro, Mass.), Celgard™ membranes (Separations ProductsDivision, Hoechst Celanese Corp., Summit, N.J.), Sartorious membranes(#11606, Sartorious Corp., Edgewood, N.Y.), Millipore membranes(Millipore Corp., Bedford, Mass.) or other membranes having the desiredpermeability properties (e.g., molecular weight exclusion limits forsolutes, particle size exclusion limits for suspended particles, chargeproperties, hydrophobicity, hydrophilicity, tensile strength, chemicalresistance, thermal resistance, etc.) according to the particularconditions to be employed. Such membranes may also in certainembodiments provide an aseptic barrier between the electrodes, which maynot exist in a sterile environment, and the biological source cell inliquid medium which typically may be desirably maintained as a sterilesuspension.

[0031] Whereas it may be particularly preferred to employ membranes thatare situated between electrode and biological source cell, as notedabove, the present invention need not be so limited and contemplatescertain embodiments wherein such membranes may not be present. Thesuitability of the bioreactor configuration (i.e., with or without amembrane protecting the electrode) may therefore depend in part on avariety of factors, including the nature of the biological source cell,the composition of the liquid medium, the composition of the bioreactoritself including the electrode composition, the waveform regulatedelectric field potential, the duration of the biomass production, or anynumber of other factors that will be appreciated by the ordinarilyskilled artisan. For example, in certain such embodiments bioreactorconfigurations may be amenable to aquaculture and/or plant hydroponiccultures, whereby the positioning of one or more electrodes within abioreactor may be accomplished without the use of a permeable membrane.

[0032] The separate liquid-medium reservoir comprises a closed tankhaving liquid medium therein, a circulating means for circulating liquidmedium between the liquid-medium circulation chamber and the separateliquid-medium reservoir, and an ion trap in fluid communication with theinterior of the closed tank. According to this embodiment, liquid mediumis circulated between (i) the liquid-medium circulation chamber immersedin the liquid medium within the liquid-medium containment vessel of thebioreactor, and (ii) the separate liquid-medium reservoir in a mannerthat permits trapping of at least one ion from the liquid medium in theion trap.

[0033] The ion trap may be any device, apparatus, column, chamber,filter, cartridge, matrix or the like with which the liquid medium mayretrievably come into contact such that at least one ion, for instance,an electrolytic by-product generated by application of awaveform-regulated electric field potential, is removed from the liquidmedium. Liquid medium so depleted of at least one ion is then returnedto the circulation chamber by the circulating means. In a preferredembodiment, the ion trap removes ions from the liquid medium byattraction of the ions to be removed to an electrical charge that ismaintained at the surface of the ion trap that contacts the liquidmedium.

[0034] For example, the ion trap may comprise an electrode connected toan electrical current source and constructed of a conductive materialthat is compatible with the liquid medium. Preferably, such an ion trapelectrode is configured to have a large surface area relative to theliquid medium volume that may contact such an electrode at any giventime. For instance, a conductive metal mesh or screen or a perforatedmetal sheet or the like may provide a useful electrode for an electrodeion trap. Typically, part or all of the electrode contact surfaceprovides a region for enhanced removal of ions from the liquid medium,which in certain embodiments may be undesirable by-products that mayadversely affect biomass production, and in other embodiments may beusefully recovered products. In certain embodiments of the invention,the ion trap electrode is used in conjunction with a waveform-regulatedelectric field potential, which may be regulated by the same waveformthat regulates the potential applied to the liquid-medium containmentvessel electrodes or may be an independent waveform. In certain otherembodiments the ion trap electrode may be connected to, or a part of, aliquid-medium containment vessel electrode.

[0035] Alternatively, the ion trap may employ chemical rather thanelectrical charge to remove ions from the liquid medium. Such a chemicalion trap, for example, may take the form of a solid-phase ion exchangemedium and, depending upon the ion exchange medium selected, may providethe further advantage of ion selectivity. Numerous electrode materialsand chemical ion exchange materials are known to those familiar with theart and can be selected in view of the teachings herein.

[0036] By way of non-limiting theory, sequestration of the electrodewithin the liquid-medium circulation chamber prevents direct contact ofthe biological source cell with the electrode, thereby reducing oreliminating electrode fouling. This configuration also permits appliedfield potentials to migrate through the entire liquid medium contents ofthe liquid-medium containment vessel by virtue of their passage throughthe permeable membrane wall of the circulation chamber into the regionof the containment vessel occupied by the biological source cell. Thecirculation of liquid medium between the circulation chamber and theseparate reservoir further permits removal from the containment vesselof undesirable contaminants that may otherwise accumulate in thevicinity of the electrode, for example, certain ionized speciesgenerated by the waveform regulated electric field potential that mayhave deleterious effects on volumetric productivity, including reactivefree radicals, noxious or toxic gases or other species.

[0037] It will be appreciated that the circulation means may takevarious forms, depending on the configuration of the liquid-mediumcontainment vessel, the circulation chamber, the separate liquid-mediumreservoir and the ion trap, so long as medium is removed from thecirculation chamber (e.g., contaminated medium is removed from thevicinity of the electrode) to the reservoir and replaced with acomparable volume of medium from the reservoir (e.g., decontaminated orfresh medium). Moreover, the circulation means may be located anywherewithin the bioreactor provided it is capable of creating a suitablemotive force within the liquid medium to ensure continual withdrawal ofmedium from the circulation chamber and replacement of such withdrawnmedium with a substantially equivalent volume of medium from theliquid-medium reservoir. In certain embodiments the circulation meansmay be provided by the configuration of the liquid-containing componentsof the bioreactor alone or in concert with mixing effects produced byone or more of ion injection, ion irrigation, ion migration or otherelectrohydrodynamic effects provided by the apparatus and method of thepresent invention. In certain other embodiments, the circulation meansmay be provided by a pump, a turbine, a motor, a hydraulic device, ahydrostatic, gravitational or capillary-driven device, or any othersuitable device for circulating liquid medium as provided herein andwith which those having ordinary skill in the art will be familiar.

[0038] In certain other embodiments, the bioreactor may include in theliquid-medium containment vessel two liquid-medium circulation chambersas described above, wherein the first electrode is positioned within afirst liquid-medium circulation chamber that is in fluid communicationwith a first separate liquid-medium reservoir comprising a first iontrap, and the second electrode is positioned within a secondliquid-medium circulation chamber that is in fluid communication with asecond separate liquid-medium reservoir comprising a second ion trap.For example, according to one such embodiment the first electrode is ananode and the second electrode is a cathode, and the selectivities ofthe first and second ion traps may be for distinct ionic species. Asanother example, in certain such embodiments the liquid medium deliveredby at least one circulating means to at least one circulating chambermay be fresh medium from the separate reservoir, or may be mediumenriched in a specific medium component or deficient in a specificmedium component. Numerous variations in the materials, devices andmethods of this invention, within the scope of the appended claims, willoccur to those skilled in the art in light of the present disclosure.

[0039] Biological Source

[0040] A biological source cell may comprise any cell or tissuepreparation derived from a biological source in which viable andpreferably intact cells are present. In most preferred embodimentsbiological source cells are present in suspension in liquid medium,either as single cells or as aggregates which may be homogeneous or maybe heterogeneous with respect to cell type. Biological source cells maybe provided, for example, by obtaining a characterized oruncharacterized specimen from a laboratory, a clinic or from the field,or by obtaining a tissue explant, organ culture, blood sample, biopsyspecimen or any other tissue or cell preparation from a biologicalsource. As noted above, in certain embodiments biological source cellsmay be present in the form of plants or plant tissues, such as in ahydroponic bioreactor configuration, or in the form of other biologicalmaterial, for example, in aquaculture.

[0041] The biological source may be a prokaryote, for example abacterium such as Escherica coli, Staphylococcus aureus, Pseudomonasaeruginosa or Bacillus thuringiensis. The biological source may also bean archaebacterium, for example, Marinococcus or Sulfolobus shibatae.The biological source may also be a eukaryote, such as a yeast, afungus, a plant, an invertebrate animal or a vertebrate animal,including a human or non-human vertebrate. Examples of biologicalsources that are yeasts include Phaffia rhodozyma (source of the widelyused pigment astaxanthin), Saccharomyces cerevisiae, Schizosaccharomycespombe, Pichia pastoris, Pichia stipitis, Candida utilis, Candidaalbicans, Candida guilliermondii and Cryptococcus albidus.

[0042] Biological sources that are fungi for use according to thepresent invention include, for example, Metarhizium flavivoride,Beauvueria bassiana, Paecilomyces fumosoreus, Gladiocladium fimbriatumand other fungi for which enhanced biomass production may be desirable.Such fungi include, for example, edible mushroom species and fungalspecies that specifically infect one or more insect species, and thusmay provide useful vectors for specific biocides such as insecticides.Biological sources that are plants for use according to the presentinvention include any of numerous plant species that provide cellsamenable to culturing as provided herein, for example, Taxus brevifolia(i.e., Pacific yew, the source of Taxol).

[0043] Other biological sources for use according to the presentinvention include invertebrate animals, for example, nematodes (e.g., C.elegans), insects (e.g., Spodoptera frugiperda, Estigmene acrea orTrichoplusia ni) and other invertebrates. A biological source that is avertebrate animal for use according to the present invention may be areptile, an amphibian, a bird, a fish or a mammal. In certain preferredembodiments, the biological source may be a mammal, such as a human, anon-human primate, a rodent, a bovine, an equine, an ovine or a porcineanimal. Thus, according to the present invention, a biological sourcefrom which a biological source cell may be derived includes a human ornon-human animal, a primary cell culture or culture adapted cell lineincluding but not limited to genetically engineered cell lines that maycontain chromosomally integrated or episomal recombinant nucleic acidsequences, immortalized or immortalizable cell lines, somatic cellhybrid or cytoplasmic hybrid cell lines, differentiated ordifferentiatable cell lines, transformed cell lines and the like.Numerous biological source cells are known to those having ordinaryskill in the art and are available from a variety of sources, including,for example, the American Type Culture Collection (ATCC, Manassas, Va.).

[0044] Waveform Regulated Electric Field Potential

[0045] According to the present invention, there are provided anapparatus and processes for enhancing biomass production by ioninjection into a liquid medium containing a biological source cell in abioreactor, and by ion migration through the liquid medium. The presentinvention is directed in part to waveform regulated electric fieldpotentials (i.e. alternating or direct current) whereby a repeatingwaveform governs the magnitude of an applied field over time, thewaveform having two or more modes that order the application of fieldpotentials to optimize volumetric productivity. A first waveform modegenerates an ion from an ionizable component of the liquid medium, and asecond waveform mode induces migration of the ion in the liquid medium.Optionally, a plurality of similar or dissimilar waveform modes may beincluded within a waveform according to the methods of the presentinvention. The present disclosure also provides the unexpected findingthat an optional lag phase (e.g., a period of reduced or no appliedelectric field potential) between such first and second waveform modes(and potentially between others of the plurality of modes that maycomprise the waveform) may further optimize volumetric productivity,where such a lag phase may be a function of one or more variable factorsto which the present invention pertains, including the selection ofbiological source cell and liquid medium, the size and geometricconfiguration of the liquid-medium containment vessel of the bioreactor,the applied electric field potentials and possibly other factors.

[0046] Ion injection may be achieved by initiating the generation of atleast one ionic species at electrode surfaces through application of anelectric field potential, wherein the field potential is a voltage pulseregulated by electrical waveform shaping. For example, to provide ioninjection in particularly preferred embodiments of the invention,positive and/or negative waveform induced electrodes may be sites forgeneration of monatomic oxygen (O⁻) anions in an aqueous liquid mediumby electrolytic dissociation of water. Depending on the biologicalsource cell, the liquid medium and the waveform, other gaseous ions maybe generated to enhance biomass production, for example CO₂, ammonium,nitrate or other ions.

[0047] Accordingly, ion injection provided by applying a waveformregulated electric field potential obviates the need for bubbling gasthrough liquid media in bioreactors (sparging), or for employing airliftdevices in bioreactors, and thus avoids the mechanical damage (e.g.,shearing) to cells associated with sparging or airlift mixing inbioreactors. Ion migration (also referred to as ion irrigation) withinliquid medium in a bioreactor may also be achieved through applicationof a distinct waveform mode field potential, whereby ions generated byion injection are induced to migrate within the applied field. Thus, thevoltage used to induce migration of the ions produced by the initialpulse (e.g., first waveform mode), and the duration of the subsequentapplied migration voltage (e.g., second waveform mode) provideelectrically induced diffusion of one or more desired ionic speciesthroughout the liquid medium, where availability of such ions to thebiological source cells promotes biomass production.

[0048] Variations in the particular waveform selected will be determinedin part by the particular bioreactor, biological source cell and liquidmedium that are employed. As a further variation, the waveform may incertain embodiments be applied alternatingly with forward and reversedpolarity to the bioreactor electrodes, to enhance theelectrohydrodynamic mixing of ions generated and migrated in the liquidmedium by the waveform regulated electric field potential. For example,such electrohydrodynamic mixing may be enhanced by including in theliquid medium a stable, charged suspension particle having a buoyantdensity at or near that of the liquid medium, such that alternatingreversals of electrode polarity may effect particle movement within themedium. In still other embodiments, the waveform itself may be variedduring the course of a bioreactor production run, to alter distributionof ions within the medium. Thus, in addition to providing a waveform forenhancing biomass production by promoting cell growth, the presentinvention also contemplates, for example, a variant waveform thatpromotes separations of ionic species within liquid medium in thebioreactor. Various modifications of bioreactor configuration,components and contents may be considered within these contemplatedembodiments, which may include, for example by way of illustration andnot limitation, waveform-induced electrophoretic (or, e.g.,isotachophoretic) migration of reactants and/or products within aliquid-medium bioreactor, or other separation techniques that may relateto molecular separations based on electrostatic properties.

[0049] Selection of a suitable waveform may be according to any of avariety of methods known in the art and based on the teachings providedherein. In preferred embodiments the waveform is regulated by a computerthat can be programmed to control the electric field potential output ofa suitable power source such as an AC or a DC power supply. As is wellknown in the art, such power supplies can be programmed to deliver anelectric field potential, for example, as constant voltage (V), constantcurrent (I) or constant power (W=I²R). In preferred embodimentsaccording to the present invention, field potentials are delivered atconstant current with voltage drops varying according to the particularwaveform, but the invention need not be so limited. For example, one ormore additional parameters (e.g., pH; electrochemical changes such asconductivity, resistance or capacitance; oxygen generation, etc.) may bemonitored through the use of appropriate sensors optionally in contactwith liquid medium at one or more locations within the bioreactor, suchthat feedback protocols that may modify the waveform can be activated.As noted above, the amount of voltage applied during each mode of thewaveform, and the duration of each mode, influence the ionization ofmedium components and the diffusion or migration of ions so generated.In certain embodiments, the invention provides a method for purifying,isolating, removing, segregating, enriching or otherwise accumulatingparticular biomass products (e.g., liquid medium solutes such asproteins, peptides, lipids, nucleic acids, carbohydrates, metabolites,catabolites and the like; or liquid medium particles including cells,organelles, inclusion bodies, aggregates and the like; or otherproducts) by selection of a waveform that orders a regulated fieldpotential such that the desired products migrate to a specific zone orregion within the bioreactor, from which they can be collected.

[0050] Waveforms that are suitable for a particular set of biologicalsource cell growth conditions may be identified through the use ofwaveform generating software combined with optimization of parameters asprovided herein. Potential drops required for electrolytic dissociationof ionizable species in solution are known for many ionizable componentsthat are typically components of liquid media as described above and inthe Examples, and methods for empirical determination of such potentialsare well known in the art (see, e.g., CRC Handbook of Chemistry andPhysics, CRC Press, Boca Raton, Fla.). For example, electrolyticdissociation of oxygen anion from water requires a potential of 12.3 V,while electrolytic dissociation of other ions from non-covalentcomplexes that may typically be components of liquid media (e.g., Na⁺,K⁺, Cl⁻) may require an applied field on the order of less than 0.5-1 V.Waveform-generating software may be employed and one or more parametersof volumetric productivity or other bioreactor solution conditions(e.g., cell density, pH, oxygen or CO₂ content, temperature, opticaldensity, turbidity, viscosity, conductivity, dielectric constant, etc.)may be monitored, in order to optimize selection of a suitable waveformfor enhanced biomass production using a particular biological sourcecell, liquid medium and bioreactor. Alternatively, an electricfield-optimizing computer program, for example INFOLYTICA software(Infolytica Ltd., Montreal, Canada, www.infolytic.com) can be employed,whereby real-time multiple parameter monitoring of conditions at one ormore discrete locations within the bioreactor can be conducted toidentify the relative effects of different waveforms These and relatedapproaches to selecting a suitable waveform will be apparent to thosefamiliar with the art, based on the teachings herein.

[0051] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

[0052] The following Example illustrates the invention and is notintended to limit the same. Those skilled in the art will recognize, orbe able to ascertain through routine experimentation, numerousequivalents to the specific substances and procedures described herein.Such equivalents are considered to be within the scope of the presentinvention.

EXAMPLES Example 1

[0053] Enhanced Growth of Saccharomyces cerevisiae in a Liquid-MediumBioreactor with Electrical Waveform Shaping

[0054]Saccharomyces cerevisiae (strain 1098) inoculum was obtained fromcommercial sources and cultures were initiated using standard methods.Control and experimental cylindrical glass liquid-mediumbioreactor-fermentors (length 14 inches, diameter 6 inches, fitted ateither end with stainless steel mesh electrodes) were inoculated withlog phase cultures to a final concentration of 10⁴ per milliliter. Themedium used was standardized per published protocols for S. cerevisiae(1% w/v yeast extract, 2% w/v tryptone peptone, 2% w/v dextrose).Following inoculation, the bioreactors were interconnected and thevessels were cross-circulated for six hours prior to the initiation ofquantitative analysis to ensure homogeneous starting parameters. At sixhours post-inoculation, cell counts were taken for confirmation and therun was started, with the experimental bioreactor (but not the control)continuously receiving the waveform-regulated applied electric fieldpotential depicted in FIG. 4. Cell counts, pH, and temperature readingswere taken at six hour intervals beginning 18 or 24 hourspost-inoculation. Results are shown in FIG. 3, wherein cell counts fromthe control bioreactor (circles) and cell counts from the experimentalbioreactor exposed to the waveform-regulated applied electric fieldpotential (triangles) were plotted as a function of time.

[0055] From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. A method for enhancing biomass production in abioreactor, comprising: providing a liquid-medium bioreactor thatcomprises (a) a liquid-medium containment vessel having an axis andcomprising a wall and a floor and containing a liquid medium, and (b) afirst and a second electrode mounted on said wall and mutually opposedalong said axis of the liquid-medium containment vessel and immersed inthe liquid medium, wherein (i) the first electrode comprises a firstelectrode surface and the second electrode comprises a second electrodesurface, and said first and second electrode surfaces are substantiallyparallel to one another, and (ii) the first and second electrodes areinsulated from said wall and said floor of the containment vessel,wherein said liquid medium comprises at least one ionizable component;and applying a waveform-regulated electric field potential to at leastone biological source cell within the liquid medium in the liquid-mediumbioreactor, wherein said waveform regulated electric field potentialcomprises a first waveform mode field potential capable of generating atleast one ion from the ionizable component when applied to the liquidmedium, and a second waveform mode field potential capable of inducingmigration of said ion within the liquid medium when said second waveformmode field potential is applied to the liquid medium, and therebyenhancing biomass production in the bioreactor.
 2. The method of claim 1wherein: (a) the liquid-medium containment vessel farther comprises atleast one liquid-medium circulation chamber immersed in the liquidmedium, said circulation chamber being in fluid communication with aseparate liquid-medium reservoir; (b) at least one electrode of theliquid-medium containment vessel is positioned within the liquid-mediumcirculation chamber, said liquid-medium circulation chamber comprising(i) a continuous permeable membrane wall situated between the electrodeand the biological source cell, and (ii) a conduit to said separateliquid-medium reservoir; and (c) said separate liquid-medium reservoircomprises (i) a closed tank having liquid medium therein, (ii) acirculating means for circulating liquid medium between theliquid-medium circulation chamber and the separate liquid-mediumreservoir, and (iii) an ion trap in fluid communication with theinterior of the tank; whereby circulation of liquid medium between theliquid-medium circulation chamber and the separate liquid-mediumreservoir permits trapping of at least one ion from the liquid medium inthe ion trap.
 3. The method of claim 2 wherein (a) the first electrodeis positioned within a first liquid-medium circulation chamber that isin fluid communication with a first separate liquid-medium reservoircomprising a first ion trap, and (b) the second electrode is positionedwithin a second liquid-medium circulation chamber that is in fluidcommunication with a second separate liquid-medium reservoir comprisinga second ion trap, whereby circulation of liquid medium between thefirst liquid-medium circulation chamber and the first separateliquid-medium reservoir permits trapping of at least one first speciesof ion from the liquid medium in the first ion trap and circulation ofliquid medium between the second liquid-medium circulation chamber andthe second separate liquid-medium reservoir permits trapping of at leastone second species of ion from the liquid medium in the second ion trap.4. The method of claim 1 wherein the waveform regulated electric fieldpotential is bimodal.
 5. The method of claim 1 wherein the waveformregulated electric field potential comprises at least three modes. 6.The method of claim 1 wherein the biological source is selected from thegroup consisting of a prokaryote, an archaebacterium and a eukaryote. 7.The method of claim 6 wherein the prokaryote is selected from the groupconsisting of Escherica coli, Staphylococcus aureus, Pseudomonasaeruginosa, and Bacillus thuringiensis.
 8. The method of claim 6 whereinthe eukaryote is selected from the group consisting of a yeast, afungus, a plant, an invertebrate animal and a vertebrate animal.
 9. Themethod of claim 8 wherein the yeast is selected from the groupconsisting of Phaffia rhodozyma, Saccharomyces cerevisiae,Schizosaccharomyces pombe, Pichia pastoris, Pichia stipitis, Candidautilis, Candida albicans, Candida guilliermondii and Cryptococcusalbidus.
 10. The method of claim 8 wherein the fungus is selected fromthe group consisting of Metarhizium flavivoride, Beauvueria bassiana,Paecilomyces fumosoreus and Gladiocladium fimbriatum.
 11. The method ofclaim 8 wherein the plant is Taxus brevifolia.
 12. The method of claim 8wherein the invertebrate animal is selected from the group consisting ofa nematode and an insect.
 13. The method of claim 8 wherein thevertebrate animal is selected from the group consisting of a reptile, anamphibian, a bird, a fish and a mammal.
 14. The method of claim 13wherein the mammal is selected from the group consisting of a human, anon-human primate, a rodent, a bovine, an equine, an ovine and aporcine.
 15. The method of claim 6 wherein the archaebacterium isselected from the group consisting of Marinococcus and Sulfolobusshibatae.
 16. The method of claim 1 wherein the liquid medium is anaqueous medium.
 17. The method of claim 1 wherein the liquid mediumcomprises 1% yeast extract, 2% tryptone peptone and 2% dextrose.
 18. Themethod of claim 1 wherein the ionizable component is water.
 19. Themethod of claim 1 wherein the ionizable component is an organic moleculehaving a hydroxyl group.
 20. The method of claim 1 wherein the ion isselected from the group consisting of singlet oxygen, Cl⁻, Na⁺, K⁺ andammonium.
 21. The method of claim 1 wherein the first waveform modefield potential is at least 12.3 volts.
 22. The method of claim 1wherein the second mode field potential is not greater than one volt.23. A method for enhancing biomass production in a bioreactor,comprising: A. providing a liquid-medium bioreactor that comprises (1) aliquid-medium containment vessel having an axis and comprising a walland a floor and containing a liquid medium, and (2) a first and a secondelectrode mounted on said wall and mutually opposed along said axis ofthe liquid-medium containment vessel and immersed in the liquid medium,wherein (a) the first electrode comprises a first electrode surface andthe second electrode comprises a second electrode surface, and saidfirst and second electrode surfaces are substantially parallel to oneanother, and (b) the first and second electrodes are insulated from saidwall and said floor of the containment vessel, (c) said liquid mediumcomprises at least one ionizable component, (d) the liquid-mediumcontainment vessel comprises at least one liquid-medium circulationchamber immersed in the liquid medium, said circulation chamber being influid communication with a separate liquid-medium reservoir, (e) atleast one electrode of the liquid-medium containment vessel ispositioned within the liquid-medium circulation chamber, saidliquid-medium circulation chamber comprising (i) a continuous permeablemembrane wall situated between the electrode and the biological sourcecell, and (ii) a conduit to said separate liquid-medium reservoir, and(f) said separate liquid-medium reservoir comprises (i) a closed tankhaving liquid medium therein, (ii) a circulating means for circulatingliquid medium between the liquid-medium circulation chamber and theseparate liquid-medium reservoir, and (iii) an ion trap in fluidcommunication with the interior of the tank, whereby circulation ofliquid medium between the liquid-medium circulation chamber and theseparate liquid-medium reservoir permits trapping of at least one ionfrom the liquid medium in the ion trap; and B. applying awaveform-regulated electric field potential to at least one biologicalsource cell within the liquid medium in the liquid-medium bioreactor,wherein said waveform regulated electric field potential comprises afirst waveform mode field potential capable of generating at least oneion from the ionizable component when applied to the liquid medium, anda second waveform mode field potential capable of inducing migration ofsaid ion within the liquid medium when said second waveform mode fieldpotential is applied to the liquid medium, and thereby enhancing biomassproduction in the bioreactor.